Trichomonas vaginalis in bronchoalveolar lavage fluid of a patient with severe pneumonia detected by metagenomic next-generation sequencing: A case report

Rationale: Trichomonas vaginalis (T. vaginalis) is a common anaerobic parasitic protozoan. However, to the best of our knowledge, there are few reports documenting T. vaginalis infection outside the genitourinary tract. Severe pneumonia caused by T. vaginalis infection has been rarely reported. Patient concerns: The 80-year-old female patient had a 20-year history of type II diabetes; however, she was not on regular medication. She was hospitalized due to a coma which continued 2 hours caused by trauma after a car accident. After her admission, she was provided with continuous mechanical ventilation; during the ventilation, she was still in a coma, accompanied by repeated fever and presence of much yellow sticky phlegm. The head CT scan indicated temporal lobe hematoma and subarachnoid hemorrhage. The lung CT scan showed bilateral pulmonary inflammatory consolidation and mass lesions. Diagnoses: She was initially diagnosed with severe pneumonia and acute respiratory distress syndrome. Subsequently, fiberoptic bronchoscopy was conducted, and bronchoalveolar lavage fluid (BALF) was collected and sent for metagenomic next-generation sequencing (mNGS). The result indicated the presence of abundant sequences from the T. vaginalis genome. Thus, she was diagnosed with pulmonary T. vaginalis infection. Intervention: Anti-infective ornidazole treatment has significantly improved her symptoms. Outcomes: After treatment, the patient regained consciousness and was able to communicate, and there was no obvious expectoration, fever, or positive bronchus sign in the lungs. Thereby, she was discharged from the hospital. Lessons: Special attention should be paid to infections other than common bacterial infections, such as T. vaginalis. Moreover, infection of rare pathogenic microorganisms might show symptoms similar to common bacterial infection, leading to misdiagnosis, further highlighting the usefulness of mNGS in detecting pathogens in a timely, sensitive, and accurate manner.


Introduction
Trichomonas vaginalis (T.vaginalis) is a common anaerobic parasitic protozoan that has caused non-viral sexually transmitted infection worldwide. [1]Patients infected with T. vaginalis will typically show various symptoms, including vaginal discharge, vaginitis, pruritus, and/or dysuria. [2,3]However, some infected patients do not present typical symptoms.Persistent T. vaginalis infections might cause infertility or unfavorable birth outcomes. [4]Moreover, T. vaginalis infection is associated with an elevated risk of human immunodeficiency virus infection and cervical cancer development. [5,6]However, to the best of our knowledge, there are few documented cases concerning Trichomonad infection in body systems beyond the genito-urinary tract, as T. vaginalis has a preference for inhabiting the

The study was supported by project "Exploration the clinical value of respiratory etiology BALF gene test in pulmonary infection" from Cangzhou Municipal Science and Technology Bureau (NO. 222106059).
Written informed consent was obtained from the next of kin for the patient, regarding the publication of this case report.

The authors have no conflicts of interest to disclose.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
This study was performed in accordance with the Helsinki Declaration and approved by the Ethics Committee of the Cangzhou People Hospital (No. AF/ SC-08/02.0).genitourinary tract and demonstrates specificity for this particular site. [7]ulmonary T. vaginalis infections in adults and children are relatively rare.In fact, pulmonary Trichomonas tenax infection has been found mainly in patients with cancer or other lung diseases. [8]For example, Leterrier et al reported the detection of Trichomonas tenax in the pleural fluid of a glioblastoma patient with severe pleurisy. [9]As other examples, pulmonary T. vaginalis infection has been reported in neonates and adults with respiratory distress. [10,11]Nevertheless, to the best of our knowledge, death caused by pulmonary T. vaginalis infection has rarely been reported. [12]Meanwhile, various trichomonas species in clinical cases bring great challenges in the diagnosis of related infectious diseases.
With the rapid development of next-generation sequencing (NGS), DNA or RNA sequencing has become an accurate and effective method for the detection of pathogens. [13]In clinical practice, many rare pathogens in various critical infectious diseases have been identified using NGS.In a recent case presented as severe diffuse alveolar hemorrhage, metagenomic NGS (mNGS) has been proven to be a powerful tool to identify the pathogen (i.e., leptospira) in the bronchoalveolar lavage fluid (BALF). [14]As far as we know, there is currently no report of severe pneumonia caused by pulmonary T. vaginalis infection.
The current case report not only provides more reference information for the differential diagnosis of a severe pneumonia case caused by a rare pulmonary T. vaginalis infection but also showcases the power of mNGS in clinical applications, especially in identifying atypical pathogens.

Case report
On October 26, 2021, an 80-year-old woman was admitted to the emergency department of our hospital due to being in a coma for 2 hours, caused by trauma after a car accident.Written informed consent was obtained from the next of kin for the patient.This study was performed in accordance with the Helsinki Declaration and approved by the Ethics Committee of the Cangzhou People Hospital (No. AF/SC-08/02.0).The car accident caused multiple head and limb injuries, unconsciousness, and vomiting in the patients.The head computed tomography (CT) scan indicated temporal lobe hematoma and subarachnoid hemorrhage.The joint CT scan showed multiple fractures on the right tibia and fibula.Subsequently, the subdural hemorrhage and necrotic brain tissues were removed, and an open reduction of fracture was conducted.The patient was provided with continuous mechanical ventilation during the operation, but she remained in a coma after the operation, accompanied by repeated high fever and presence of yellow sticky phlegm.The lung CT scan suggested inflammatory consolidation and mass lesions in bilateral lungs.
The patient had a 20-year history of type II diabetes and had an unstable blood sugar level, but she was not on regular medication.Moreover, she had a 20-year history of coronary atherosclerotic heart disease and took intermittent Suxiao Jiuxin Pills (i.e., a Chinese patent drug mainly composed of Chuan Xiong (Rhizoma Chuanxiong) and Bing Pian (Borneolum), at 40 mg/ tablet, produced by Tianjin Zhongxin Pharmaceutical Inc. (Tianjin, China)), which are a quick-acting heart reliever.She had no history of hypertension, infectious disease, or allergy, and no previous personal or family history of T. vaginalis infection.
After admission, the patient was in a light coma with a Glasgow coma scale of 6, and the oxygen saturation level was 96%, with a mechanical ventilation fraction of inspired oxygen (FiO2) of 60%.Both light and corneal reflexes were sluggish.The right ankle was swollen and deformed, and the bilateral Babinski signs were positive.There were rough sound of breath, wheezing, and coarse rales in bilateral lungs.The white blood cell count was 11.09 × 10 9 /L, neutrophil ratio was 95.4%, hemoglobin level was 100 g/L, and platelet (PLT) count was 164 × 10 9 /L.C-reactive protein level was 219.50 mg/L, procalcitonin level was 1.378 ng/mL, and oxygenation index was 250.Sputum culture indicated Pseudomonas aeruginosa infection, and the conventional bacterial culture of BALF was negative.No abnormality was observed by gynecological ultrasound and abdominal and pelvic CT examinations.However, a CT scan of the lungs indicated bilateral pneumonia, enlarged pulmonary artery, and left pleural effusion in the patient (Fig. 1).
As she remained unconscious and had yellow sticky phlegm, we performed a tracheotomy and maintained continuous mechanical ventilation.Meanwhile, intermittent tracheoscopy was performed to facilitate airway management to promote sputum drainage and maintain the patency of the airway.Meropenem was used for anti-infective therapy, but repeated fever (39.0˚C) and the presence of yellow sticky sputum had not been improved significantly.Then, BALF was sent for mNGS; after a strict sequence alignment with the help of a microbiologist, fragments of T. vaginalis, Candida glabrata, Atopobium vaginae, and Gardnerella vaginalis genomes were identified.The number of detected T. vaginalis sequences was 5335.Thus, the patient was diagnosed with severe pneumonia, pulmonary T. vaginalis infection, acute respiratory distress syndrome, brain contusion, and traumatic subarachnoid hemorrhage.
Previous empirical broad-spectrum antibiotics (Meropenem) treatment showed no effects; however, after the detection of T. vaginalis by mNGS, we applied ornidazole and the patient condition was significantly improved.Specifically, after 35 days of treatment with ornidazole injection (once every 12 hours, from November 19, 2021 to December 24, 2021), the patient regained consciousness and was able to communicate, and there was no obvious expectoration, fever, or positive bronchus sign in the lungs.The lung CT scan showed that inflammatory exudation and solid lesions almost completely disappeared (Fig. 2, on December 25, 2021).Thus, the patient was discharged from the hospital.

Discussion and conclusions
In this case report, the patient was hospitalized due to a 2-hour coma caused by trauma after a car accident.She was diagnosed with severe pneumonia and pulmonary T. vaginalis infection.It is extremely rare that T. vaginalis was detected in BALF.Further, the detection of T. vaginalis in the lungs indicated that more attention should be paid to rare pathogenic microorganisms in critical diseases.
The patient head CT scan indicated temporal lobe hematoma and subarachnoid hemorrhage, and the lung CT scan indicated inflammatory consolidation and mass lesions in bilateral lungs.Accordingly, severe pneumonia and acute respiratory distress syndrome were considered; however, little improvement was observed after treatment with anti-infective therapy.The BALF was sent for mNGS, and there was a high load of T. vaginalis sequences, suggesting the patient had a pulmonary T. vaginalis infection.Actually, the symptoms and the common physical, laboratory and imaging examination results both suggested bacterial pneumonia.However, since there was no response to empirical broad-spectrum antibiotics (meropenem), and mNGS suggested a T. vaginalis infection, ornidazole was started and the patient condition was significantly improved.
As a common parasitic protozoan colonized in the urogenital tract, T. vaginalis has been rarely detected outside the genitourinary tract, such as causing severe pneumonia in the lungs. [15]It has been demonstrated that Trichomonas tenax (another species of Trichomonas) mainly causes bronchopulmonary infections in patients with cancer or other lung diseases. [8]However, pulmonary T. vaginalis infection has never been reported in adults.In 2002, Szarka et al documented 2 cases of neonatal pneumonia in which T. vaginalis was detected in a newborn tracheal discharge, and subsequently in her mother tracheal discharge as well. [16]hese 2 newborn babies suffered from severe congenital breathing difficulties.The microscopic examination and CPLM medium culture finally confirmed the symptom was caused by T. vaginalis infection.Whereas in another more recent case (2020) of neonatal pneumonia caused by T. vaginalis, the patient also benefited from a 28-day targeted antibiotic therapy after the diagnosis of T. vaginalis infection by multiplex real-time PCR technique. [17]hus, atypical pathogen infections should be considered when the patients do not respond to conventional therapy.Moreover, Salvador-Membreve et al demonstrated that T. vaginalis could exert a cytopathic effect on lung alveolar basal carcinoma epithelial cells, [7] supporting the unfavorable impacts of T. vaginalis on lungs.T. vaginalis infection in the urogenital tract could display distinct phenotypes. [18]However, the current adult patient was an extremely rare case.On the one hand, previous reports showed that pulmonary T. vaginalis infection mainly occurred in neonates due to maternal infection.On the other hand, there was no T. vaginalis infection in this patient genitourinary tract.She showed severe pneumonia accompanied by repeated fever and presence of much yellow sticky phlegm.This case thus provides valuable reference information on pulmonary T. vaginalis infection.Besides, it is quite hard to identify T. vaginalis infection without mNGS in this case, highlighting the significance of mNGS as a diagnostic tool for infectious diseases.
Typically, culture-based tests or immunological assays can only detect known pathogens; besides, obvious limitations exist in these detection methods, such as testing time and lack of sensitivity. [19]In contrast, the rapid development of molecular and mNGS techniques has provided more alternatives for clinical diagnosis and treatments. [20][23] Accumulating evidence has indicated the significant advantages of mNGS, including shorter diagnosis time, higher diagnostic sensitivity, and greater capacity in detecting pathogens of different species (e.g., bacteria, viruses, and fungi) compared with traditional methods. [24]or instance, Chen et al showed the effectiveness of mNGS in diagnosing critically ill or immunocompromised patients using BALF samples. [25]In the current case, mNGS revealed sequence fragments of the T. vaginalis, Candida glabrata, Atopobium vaginae, and Gardnerella vaginalis genomes in the BALF, suggesting infections from these microbes.The mNGS data helped us target causative microorganisms quickly, leading to appropriate intervention, which significantly alleviated the patient symptoms.Our results also highlight the power of mNGS in determining rare pathogens in clinical practice.Nevertheless, the current limitation of mNGS mainly comes from the report interpretation of mNGS data, [21] and thus this method should be combined with clinical symptoms as well as conventional methods.The precise interpretation of mNGS data would be further conducive to the application of mNGS in more clinical cases.
Although we tried to document this rare case in detail, there were still several limitations in our present study.This patient came to our attention after she was diagnosed with T. vaginalis infection, thus the clinical information was collected after that.Some clinical information was incomplete or missing, which obviously limited the comprehensive understand of this case.
In conclusion, we herein reported an extremely rare case that the patient was diagnosed with severe pneumonia caused by pulmonary T. vaginalis infection.Our case reminded us that special attention should be paid to infections other than common bacterial infections, such as T. vaginalis.Moreover, infection of rare pathogenic microorganisms might show symptoms similar to common bacterial infection, leading to misdiagnosis, further highlighting the usefulness of mNGS in detecting pathogens in a timely, sensitive, and accurate manner.

Figure 1 .
Figure 1.Results of CT scans of the lungs before anti-infective treatment showed the presence of bilateral pneumonia, enlarged pulmonary artery, and left pleural effusion.CT = computed tomography.